JOURNAL
OF
Vol. 32, No. 2
VIROLOGY, Nov. 1979, p. 583-592
0022-538X/79/11-0583/10$02.00/0
Salmonella Bacteriophage Glycanases: Endorhamnosidases of Salmonella typhimurium Bacteriophages STEFAN B. SVENSON,1* JORGEN LONNGREN,2 NILS CARLIN,' AND ALF A. LINDBERG' Department of Bacteriology, National Bacteriological Laboratory, S-105 21 Stockholm,' and Department of Organic Chemiistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm,2 Sweden
Received for publication 15 May 1979
Twelve bacteriophages lysing only smooth Salmonella typhimurium strains were shown to have similar morphology-an icosahedric head to which a short, noncontractile tail carrying six spikes was attached. All phages degraded their lipopolysaccharide (LPS) receptors as shown by their ability to cleave off ['4C]galactosyl-containing oligosaccharides from S. typhimurium cells labeled in their LPS. The oligosaccharides inhibited the a-D-galactosyl-specific Bandeiraea simplicifolia lectin agglutination of human type B erythrocytes, indicating that all 12 phage glycanases were of endorhamnosidase specificity, i.e., hydrolyzed the a-Lrhamnopyranosyl-(1 -- 3)-D-galactopyranosyl linkage in the S. typhimurium 0polysaccharide chain. Two of the phages, 28B and 36, were studied in more detail. Whereas the phage 28B glycanase hydrolyzed the S. typhimurium LPS into dodeca- and octasaccharides, the phage 36 glycanase in addition cleaved off tetrasaccharides. Both phage enzymes hydrolyzed the 0-polysaccharide chains of LPS from Salmonella belonging to serogroups A, B, and Dl, which are built up of tetrasaccharide-repeating units identical except for the nature of the 3,6dideoxyhexopyranosyl group (R). R al - 2)-a-D-Manp-(1 -- 4)-a-L-Rhap-(1-+ 3)-a-D-Galp-(l The phage 28B and 36 endorhamnosidases hydrolyzed also an LPS from which the 3,6-dideoxyhexosyl substituents had previously been hydrolyzed off. However, neither of the enzymes was active on LPS preparations in which the C2-C3 bond of the L-rhamnopyranosyl ring had been opened by periodate oxidation. Glucosylation at 0-6 of the D-galactopyranosyl residues in the S. typhimurium LPS was found to be incompatible with hydrolysis by both enzymes. However, in an LPS glucosylated at 0-4 of the D-galactopyranosyl residues, the adjacent a-L-rhamnopyranosyl linkages were found to be preferentially cleaved.
Receptors for several bacteriophages specific for smooth gram-negative bacteria have been shown to be located in the lipopolysaccharide (LPS) moiety of the bacterial outer membrane (12). More recently, it has been revealed that such phage adsorption often is concomitant with an enzymatic hydrolysis of the polysaccharide receptor. Thus, adsorption of phages P22 and E'5 to their LPS receptors (see Fig. 1 for general structure of Salmonella LPS) of susceptible Salmonella species is accompanied by hydrolytic cleavage of a-L-rhamnosyl linkages within the LPS (7, 11). Likewise, one Escherichia coli phage Qi8 cleaves a-D-mannopyranosyl linkages of the E. coli 0-8 LPS, and the Shigella-phage Sf6 cleaves the Shigella flexneri LPS at the aL-rhamnopyranosyl linkages (13, 17). 583
The phage glycanase activities have so far been shown to be exclusively associated with the phage tail. In some instances (P22 and coliphages f18 and 29), the phage enzymes have been purified and identified as tail structural proteins (2, 7, 17). The elucidation of properties and specificities of phage glycanases is of interest for studies of phage-receptor interactions and host specificities. Furthermore, phage glycanases may also be important as tools for specific degradation of
polysaccharides.
In a search for phages possessing glycanase activity, 12 0-antigen-specific S. typhimurium phages were found to have LPS receptor cleaving properties. In the present paper we report on the substrate specificities of two of these phage
584
SVENSON ET AL.
enzymes (no. 28B and 36) and the isolation of phage 36 subviral structures carrying glycanase
activity. MATERIALS AND METHODS Bacterial strains. S. typhimurium strains SH4305 (0-antigens 4, 5, and 122) and SH4809 (0-antigens 4, 5, and 12), S. enteritidis SH1262 (his- thr- thy-, 0antigens 9 and 12) were obtained from P. H. Miikela, Central Public Health Laboratory, Helsinki, Finland. S. paratyphi A var. durazzo (0-antigens 2 and 12), S. typhimurium LT2, and S. flexneri var. Y were from the collection at the Department of Bacteriology, National Bacteriological Laboratory, Stockholn, Sweden. Strain S. typhimurium SL3622 and the uridine diphosphate-galactose epimerase-defective strain LT2-M1 were from the collection of B. A. D. Stocker, Department of Medical Microbiology, Stanford University, Stanford, Calif. S. typhimurium strains 1, 2, 3, 4, 5a, 6a, 6b, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, and 23a were the phage typing reference strains used at the Department of Bacteriology, National Bacteriological Laboratory, Stockholm, Sweden. Bacteriophages. The clear plaque mutant P22c2 was obtained from B. A. D. Stocker. Salmonella smooth specific phages 2, 4, 8, 28B, 30, 31, 32, 33, 34, 36, 37, and 39 were those used for S. typhimurium phage typing at the Department of Bacteriology, National Bacteriological Laboratory, Stockholm, Sweden
(15).
Preparation of bacteriophage stocks. All phages were grown in submerged culture on their corresponding S. typhimurium host strain (Table 1). Log-phase cells, approximately 5 x 108 cells per ml, grown at 37°C in Marvin medium (16), were infected at a multiplicity of infection of 1 to 5. The individual cultures were heavily aerated for 5 to 6 h or until lysis was evident. Complete lysis of cells was imposed by the addition of chloroform. Cell debris was sedimented by centrifugation at 2,500 x g for 20 min. Some of the phage stocks were further purified by polyethylene glycol (PEG) precipitation. PEG (average molecular weight, 6,000) and sodium chloride were added to the clear supernatant to give final concentrations of 8% (wt/vol) and 0.5 M, respectively (23). After sedimentation for 24 h at 4°C, aggregated phages were collected by centrifugation at 4,000 x g for 20 min at 4°C. The phage pellets were suspended in M-9 base medium (1) to approximately 1/100 of the original volume, and the PEG was precipitated by repeated chloroform additions and removed by low-speed centrifugations. The resulting phage stocks had titers of 5 x 1010 to 5 x 1013 plaque-forming units (PFU) per ml. The phages 28B and 36 were in addition purified by ultracentrifugation in CsCl gradients (Spinco SW 40 rotor at 160,000 x g for 16 h). Gradients were fractionated from the bottom in about 250-Al portions, and the densities of the phage bands were determined (for 28B, 8 = 1.48; for 36, 8 = 1.49). The phage-containing fractions were finally dialyzed against M-9 base medium and titrated on their respective host strains (phage 28B, 4.7 x 10'3 PFU ml'; phage 36, 2.2 x 1013 PFU * ml-'). Isolation of phage 36 endoglycanase. A heavily aerated, 5-liter culture of logarithmically growing cells
J. VIROL.
of S. typhimurium strain 22 was infected at a multiplicity of infection of 1.5. After 5 h of propagation at 37°C, the culture was lysed by the addition of a few milliliters of chloroform and 0.5% (wt/vol) Sarkosyl. Cell debris was sedimented at 2,500 x g for 20 min at 4°C. Ammonium sulfate was added to 50% saturation of the clear supernatant, and precipitation was allowed for 24 h at 4°C. After sedimentation at 2,000 x g, the precipitate was suspended in 500 ml of phosphatebuffered saline (PBS) and dialyzed against PBS overnight. The precipitation with 50% saturated ammonium sulfate was repeated twice. After final dissolution and dialysis against PBS, the material was subjected to high-speed centrifugation to sediment remaining phage particles. The enzyme activity of the supernatant preparation was determined by its ability to release [14C]galactose-containing material from formalinized S. typhimurium LT2-M1 cells labeled in the LPS. Glycanase-active preparations were also examined by electron microscopy. Preparation of LPS. Bacteria were grown in submerged culture, and LPS was extracted by the phenolwater method from formaldehyde-killed bacteria (22). Some of the LPS preparations were made more water soluble by treatment with 0.15 M sodium hydroxide for 2 h at 1000C. This treatment hydrolyzes phosphate bonds and fatty acid ester linkages in the lipid A moiety. Preparation of abequose-deficient polysaccharide was performed by heating LPS from S. typhimurium SH4809 in 50% acetic acid at 90°C for 8 h. This treatment hydrolyzed the 2-keto-3-deoxy-D-mannooctulusonic acid linkages in the core region of the LPS as well as all a-1,3-abequosyl linkages. Sugar and methylation analyses of the dialyzed preparation showed the presence of the expected methyl ethers of D-mannose, L-rhamnose, and D-galactose. Sodium metaperiodate-sodium borohydride treatment of the S. typhimurium LPS was performed as described (8). LPS from Klebsiella 012 and the capsular polysaccharide from Klebsiella K47 were the same as used earlier (3, 6). Screening for phage glycanase activities. Screening for phage glycanase activities was performed by the method of Eriksson et al. (7). In short, mid-log-phase cells of the uridine diphosphate-galactose-4-epimeraseless mutant, S. typhimurium LT2Ml, were labeled with D-galactose-1-14C (0.5 ,uCi/ml, 2 mg/ml) in their LPS for 3 h. To measure glycanase activities, the 14C-labeled cells, twice washed in PBS, were mixed with dilutions of the phage to be tested. After 60 min of incubation at 37°C, cells were sedimented at 2,000 x g for 20 min in the cold, and portions of the supernatant were withdrawn and assayed for 14C activity. Screening for endorhamnosidase activities. To test whether a specific phage glycanase possessed the ability to cleave the a-L-rhamnosyl linkages of the S. typhimurium SH4809 LPS, the dialyzable crude oligosaccharide preparation (see below) was tested for its inhibitory activity on Bandeiraea simplicifolia lectin agglutination of human type B erythrocytes. Inhibition 50% or more of that obtained with melibiose (calculated on a molar basis assuming a mean molecular weight of the crude oligosaccharide of -1,200) was considered as positive.
S. TYPHIMURIUM PHAGE ENDORHAMNOSIDASES
VOL. 32, 1979
Electron microscopy. The preparation to be examined was stained with uranyl acetate by the following procedure: a droplet of the preparation was placed on a grid (200-mesh copper with carbon-shadowed Parlodion film), and excess fluid was sucked off with filter paper. A drop of freshly prepared 1% (wt/vol) uranyl acetate was then applied to the grid. After a few minutes, excess fluid was again removed by touching the grid to the corner of an adsorbent filter paper. The preparations were examined in a Philips EM-200 electron microscope. The estimation of phage dimensions was made by comparison with a calibration grid. Methylation analyses. Methylation analyses were performed as described earlier (14). The oligosaccharides were reduced with sodium borohydride, and after methylation and subsequent work-up, the materials were hydrolyzed. The resulting monosaccharides were transformed into alditol acetates and analyzed by gasliquid chromatography (GLC) and mass spectrometry. For gas-liquid chromatography, a Perkin-Elmer 990 instrument fitted with a 3% OV-225 colunm was used. Gas-liquid chromatography-mass spectrometry was performed with a Varian MAT 311 instrument. Test for phage glycanase hydrolysis of capsular or LPS. The phage to be assayed was mixed at a ratio of 109 to 1010 PFU/mg of polysaccharide in 5 mM ammonium carbonate buffer, pH 7.0, and incubated at 37°C within a dialysis bag immersed in 10 times the volume of the same buffer. After 48 h of incubation, the surrounding dialysis fluid was concentrated to dryness and heated to 50°C under vacuum to remove the remaining ammonium carbonate. The amount of hexose in the reaction mixture and the surrounding
Bacteria
2
4
dialysis fluid was determined by the phenol-sulfuric acid method (5). Miscellaneous methods. Gel chromatography of oligosaccharides was performed on a column (170 by 2.5 cm) of Bio-Gel P-2 (200 to 400 mesh) eluted with water, using trichlorobutanol (0.05%) as the bacteriocidal agent. Separations were monitored by a Waters 403 refractometer, and the fractions were assayed for carbohydrate content by the phenol-sulfuric acid method. For nuclear magnetic resonance recordings, a Jeol FX-100 instrument operated in the PFT mode was used. The spectra were recorded for solutions in D20 at 850C, using extemal tetramethylsilane as standard.
RESULTS
Screening for phage glycanase activity. Twelve phages were selected from a set used for phage typing of clinical isolates of S. typhimurium (15). The lytic spectra of these phages on the reference set of S. typhimurium strains is given in Table 1. The various phages were incubated with formaldehyde-treated S. typhimurium LT2-M1 cells which had been labeled with ['4C]galactose in their LPS (7). After centrifugation, '4C-labeled material was found in all supernatants, indicating that all 12 phages possessed glycanase activities (Table 2). The same batch of ['4C]galactose-labeled LT2-M1 cells was also subjected to hydrolysis by the P22 endorhamnosidase by incubating the cells with
TABLE 1. Lytic spectra of S. typhimurium phagesa 34 33 32 31 30 28B 8
37
+
+ +
+ +
+ +
+b +
+
+
+
+
+
+
+
+
+
+
+
+
+ + + + + +
+ + + +
+ + + +
+ + + +
+
+
+
+
+
2 3 4 5a 6a 6b 7 8 9 10 11 12 13 14 15 16 18 19 20 21 22 23a
+
+
+ +
+ i
+
+b
+ + + + +
+ +
+
+
+
+b + +
+b
+ +
+
+
+ + + +
39
36
+
1
+
585
+
+
+
+ +
+ +
+
+b+
+ +
+b
+
+
+
+b
+b
+
+
+
+
+
+
+b
+b +
+
+
+
+
+
+
+ +b
+
+
+
+ +
+
+
+
a Phages investigated on the standard set of S. typhimurium strains used for phage typing of clinical isolates. +, Sensitivity to phage indicated. b Strain was used for propagation.
586
SVENSON ET AL.
TABLE 2. Glycanase activities of S. typhimurium phages assayed by ["4C]galactose release' Phage Titer % Releaseb 2 5 x 107 10 (4) 4 1.5 x 1010 49 (33) 8 3.2 x 1012 50 (48) 28B 3.1 x 1012 55 (48) 30 6.7 x 1010 43 (48) 31 1 x 109 46 (12) 32 1.6 x 1010 45 (33) 33 1.4 x 1012 64 (48) 34 1.3 x 1010 65 (32) 36 1 x 109 68 (12) 1 x 1010 37 39 (30) 1 x 109 39 54 (12) a 14C-labeled material was released from S. typhimurium LT2-M1 cells. To 100-,ul portions of washed S. typhimurium LT2-M1 cells previously labeled in their LPS with [14C]galactose were added 900-pl amounts of the various phage preparations. After incubation for 1 h at 37°C, cells were sedimented by centrifugation, 500-,ul portions of the supernatants were withdrawn, and the released 14C activity was determined. The same batch of [14C]galactose-labeled S. typhimurium LT-2 M-1 was also assayed against a PEG-purified P22c2 phage stock. Maximal release (48% of total) was obtained at 5 x 1010 PFU per assay. b Values in parentheses were obtained by using equal numbers of PFU of a purified phage P22c2 preparation.
different amounts of a PEG-purified phage P22 stock and the percentage of release compared with those obtained by the 12 phages under investigation (Table 2). In all instances (except phage 30), the crude phage lysates showed higher release activities per PFU than did the purified phage P22 stock. To confirm that the release of 14C-labeled material from the S. typhimurium LT2-M1 cells was due to phage-associated glycanase activities, crude lysates of the respective propagation strains were tested for hydrolytic properties. About 5 x 1011 cells of each strain were lysed by sonication (three times 10 s at 25W in a Labsonic 1510 sonicator), and the crude bacterial lysates were incubated with [14C]galactose-labeled S. typhimurium LT2-M1 cells. None of the bacterial lysates released any 14C-labeled material. In a control experiment, the various phage lysates were, after sonication as described above, found to be equally active as before sonication. The PEG-purified phage 28B and 36 stocks were further purified by density gradient ultracentrifugation. In both cases, the phage-containing fractions displayed the highest release activities of [14C]galactose-labeled material from S. typhimurium LT2-M1 cells. The 50% maximal release (45% of total label was maximally released) values were, for phage 28B and 36, 8.8
J. VIROL.
109 and 1.1 x 108 PFU, respectively. These data indicated that the glycanase activities of phages 28B and 36 were associated with the
x
phage particles. Screening for endorhamnosidase speci-
ficities. In another series of experiments, the various phages were incubated with partially delipidated LPS from S. typhimurium SH 4809 within dialysis sacks immersed in buffer. All 12 phages degraded the LPS to dialyzable oligosaccharides as evidenced by recovery of 30 to 50% of the total carbohydrate in the surrounding dialysis fluid (data not shown). Glycanases cleaving the a-L-rhamnosyl linkages in the S. typhimurium SH4809 LPS (Fig. 1, Table 4) should give rise to oligosaccharides possessing a terminal, nonreducing ca-D-galactosyl group. The agglutination of human type B erythrocytes with the a-D-galactosyl-specific B. simplicifolia lectin was inhibited by the crude oligosaccharide preparations (obtained from the dialysis experiments described above), thus suggesting that all 12 phages exhibited endorhamnosidase activity. Electron microscopic examination of phages. In Fig. 2, the morphology typical for all 12 phages is shown. They all have a head with hexagonal symmetry about 50 nm in diameter to which a short, noncontractile tail carrying six spikes is attached. This appearance is typical for type C phages, based on the classification of Bradley (4). Two of the phages, 28B and 36, were chosen for a more detailed study. Phage 28B was chosen for its narrow lytic spectrum (Table 1) and ease of propagation. Phage 36, however, had a broad lytic spectrum (Table 1) also lysing the S. typhimurium SL3622 (Fig. 1 and Table 4). Furthermore, phage 36 showed a high '4C release activR a
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FIG. 1. General structure of LPS from Salmonella serogroups A, B, and Dl. R indicates abequose (3,6dideoxy-D -xylo-hexose) in Salmonella B, or tyvelose (3,6-dideoxy-D -arabino-hexose) and paratose (3,6-dideoxy-D-ribo-hexose) in Salmonella Dl and A, respectively. Broken arrow indicates substitution occurring only in some strains. n, Number of repeating units in 0-chain, varying from 4 to 30.
S. TYPHIMURIUM PHAGE ENDORHAMNOSIDASES
VOL. 32, 1979
- * < =, *S t .--; '! i-
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as compared with the phage P22 standard (Table 2); i.e., it could be overproducing the glycanase. Isolation of phage 36 subviral elements possessing glycanase activity. Subviral
ity
587
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OF
Vol. 32, No. 2
VIROLOGY, Nov. 1979, p. 583-592
0022-538X/79/11-0583/10$02.00/0
Salmonella Bacteriophage Glycanases: Endorhamnosidases of Salmonella typhimurium Bacteriophages STEFAN B. SVENSON,1* JORGEN LONNGREN,2 NILS CARLIN,' AND ALF A. LINDBERG' Department of Bacteriology, National Bacteriological Laboratory, S-105 21 Stockholm,' and Department of Organic Chemiistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm,2 Sweden
Received for publication 15 May 1979
Twelve bacteriophages lysing only smooth Salmonella typhimurium strains were shown to have similar morphology-an icosahedric head to which a short, noncontractile tail carrying six spikes was attached. All phages degraded their lipopolysaccharide (LPS) receptors as shown by their ability to cleave off ['4C]galactosyl-containing oligosaccharides from S. typhimurium cells labeled in their LPS. The oligosaccharides inhibited the a-D-galactosyl-specific Bandeiraea simplicifolia lectin agglutination of human type B erythrocytes, indicating that all 12 phage glycanases were of endorhamnosidase specificity, i.e., hydrolyzed the a-Lrhamnopyranosyl-(1 -- 3)-D-galactopyranosyl linkage in the S. typhimurium 0polysaccharide chain. Two of the phages, 28B and 36, were studied in more detail. Whereas the phage 28B glycanase hydrolyzed the S. typhimurium LPS into dodeca- and octasaccharides, the phage 36 glycanase in addition cleaved off tetrasaccharides. Both phage enzymes hydrolyzed the 0-polysaccharide chains of LPS from Salmonella belonging to serogroups A, B, and Dl, which are built up of tetrasaccharide-repeating units identical except for the nature of the 3,6dideoxyhexopyranosyl group (R). R al - 2)-a-D-Manp-(1 -- 4)-a-L-Rhap-(1-+ 3)-a-D-Galp-(l The phage 28B and 36 endorhamnosidases hydrolyzed also an LPS from which the 3,6-dideoxyhexosyl substituents had previously been hydrolyzed off. However, neither of the enzymes was active on LPS preparations in which the C2-C3 bond of the L-rhamnopyranosyl ring had been opened by periodate oxidation. Glucosylation at 0-6 of the D-galactopyranosyl residues in the S. typhimurium LPS was found to be incompatible with hydrolysis by both enzymes. However, in an LPS glucosylated at 0-4 of the D-galactopyranosyl residues, the adjacent a-L-rhamnopyranosyl linkages were found to be preferentially cleaved.
Receptors for several bacteriophages specific for smooth gram-negative bacteria have been shown to be located in the lipopolysaccharide (LPS) moiety of the bacterial outer membrane (12). More recently, it has been revealed that such phage adsorption often is concomitant with an enzymatic hydrolysis of the polysaccharide receptor. Thus, adsorption of phages P22 and E'5 to their LPS receptors (see Fig. 1 for general structure of Salmonella LPS) of susceptible Salmonella species is accompanied by hydrolytic cleavage of a-L-rhamnosyl linkages within the LPS (7, 11). Likewise, one Escherichia coli phage Qi8 cleaves a-D-mannopyranosyl linkages of the E. coli 0-8 LPS, and the Shigella-phage Sf6 cleaves the Shigella flexneri LPS at the aL-rhamnopyranosyl linkages (13, 17). 583
The phage glycanase activities have so far been shown to be exclusively associated with the phage tail. In some instances (P22 and coliphages f18 and 29), the phage enzymes have been purified and identified as tail structural proteins (2, 7, 17). The elucidation of properties and specificities of phage glycanases is of interest for studies of phage-receptor interactions and host specificities. Furthermore, phage glycanases may also be important as tools for specific degradation of
polysaccharides.
In a search for phages possessing glycanase activity, 12 0-antigen-specific S. typhimurium phages were found to have LPS receptor cleaving properties. In the present paper we report on the substrate specificities of two of these phage
584
SVENSON ET AL.
enzymes (no. 28B and 36) and the isolation of phage 36 subviral structures carrying glycanase
activity. MATERIALS AND METHODS Bacterial strains. S. typhimurium strains SH4305 (0-antigens 4, 5, and 122) and SH4809 (0-antigens 4, 5, and 12), S. enteritidis SH1262 (his- thr- thy-, 0antigens 9 and 12) were obtained from P. H. Miikela, Central Public Health Laboratory, Helsinki, Finland. S. paratyphi A var. durazzo (0-antigens 2 and 12), S. typhimurium LT2, and S. flexneri var. Y were from the collection at the Department of Bacteriology, National Bacteriological Laboratory, Stockholn, Sweden. Strain S. typhimurium SL3622 and the uridine diphosphate-galactose epimerase-defective strain LT2-M1 were from the collection of B. A. D. Stocker, Department of Medical Microbiology, Stanford University, Stanford, Calif. S. typhimurium strains 1, 2, 3, 4, 5a, 6a, 6b, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, and 23a were the phage typing reference strains used at the Department of Bacteriology, National Bacteriological Laboratory, Stockholm, Sweden. Bacteriophages. The clear plaque mutant P22c2 was obtained from B. A. D. Stocker. Salmonella smooth specific phages 2, 4, 8, 28B, 30, 31, 32, 33, 34, 36, 37, and 39 were those used for S. typhimurium phage typing at the Department of Bacteriology, National Bacteriological Laboratory, Stockholm, Sweden
(15).
Preparation of bacteriophage stocks. All phages were grown in submerged culture on their corresponding S. typhimurium host strain (Table 1). Log-phase cells, approximately 5 x 108 cells per ml, grown at 37°C in Marvin medium (16), were infected at a multiplicity of infection of 1 to 5. The individual cultures were heavily aerated for 5 to 6 h or until lysis was evident. Complete lysis of cells was imposed by the addition of chloroform. Cell debris was sedimented by centrifugation at 2,500 x g for 20 min. Some of the phage stocks were further purified by polyethylene glycol (PEG) precipitation. PEG (average molecular weight, 6,000) and sodium chloride were added to the clear supernatant to give final concentrations of 8% (wt/vol) and 0.5 M, respectively (23). After sedimentation for 24 h at 4°C, aggregated phages were collected by centrifugation at 4,000 x g for 20 min at 4°C. The phage pellets were suspended in M-9 base medium (1) to approximately 1/100 of the original volume, and the PEG was precipitated by repeated chloroform additions and removed by low-speed centrifugations. The resulting phage stocks had titers of 5 x 1010 to 5 x 1013 plaque-forming units (PFU) per ml. The phages 28B and 36 were in addition purified by ultracentrifugation in CsCl gradients (Spinco SW 40 rotor at 160,000 x g for 16 h). Gradients were fractionated from the bottom in about 250-Al portions, and the densities of the phage bands were determined (for 28B, 8 = 1.48; for 36, 8 = 1.49). The phage-containing fractions were finally dialyzed against M-9 base medium and titrated on their respective host strains (phage 28B, 4.7 x 10'3 PFU ml'; phage 36, 2.2 x 1013 PFU * ml-'). Isolation of phage 36 endoglycanase. A heavily aerated, 5-liter culture of logarithmically growing cells
J. VIROL.
of S. typhimurium strain 22 was infected at a multiplicity of infection of 1.5. After 5 h of propagation at 37°C, the culture was lysed by the addition of a few milliliters of chloroform and 0.5% (wt/vol) Sarkosyl. Cell debris was sedimented at 2,500 x g for 20 min at 4°C. Ammonium sulfate was added to 50% saturation of the clear supernatant, and precipitation was allowed for 24 h at 4°C. After sedimentation at 2,000 x g, the precipitate was suspended in 500 ml of phosphatebuffered saline (PBS) and dialyzed against PBS overnight. The precipitation with 50% saturated ammonium sulfate was repeated twice. After final dissolution and dialysis against PBS, the material was subjected to high-speed centrifugation to sediment remaining phage particles. The enzyme activity of the supernatant preparation was determined by its ability to release [14C]galactose-containing material from formalinized S. typhimurium LT2-M1 cells labeled in the LPS. Glycanase-active preparations were also examined by electron microscopy. Preparation of LPS. Bacteria were grown in submerged culture, and LPS was extracted by the phenolwater method from formaldehyde-killed bacteria (22). Some of the LPS preparations were made more water soluble by treatment with 0.15 M sodium hydroxide for 2 h at 1000C. This treatment hydrolyzes phosphate bonds and fatty acid ester linkages in the lipid A moiety. Preparation of abequose-deficient polysaccharide was performed by heating LPS from S. typhimurium SH4809 in 50% acetic acid at 90°C for 8 h. This treatment hydrolyzed the 2-keto-3-deoxy-D-mannooctulusonic acid linkages in the core region of the LPS as well as all a-1,3-abequosyl linkages. Sugar and methylation analyses of the dialyzed preparation showed the presence of the expected methyl ethers of D-mannose, L-rhamnose, and D-galactose. Sodium metaperiodate-sodium borohydride treatment of the S. typhimurium LPS was performed as described (8). LPS from Klebsiella 012 and the capsular polysaccharide from Klebsiella K47 were the same as used earlier (3, 6). Screening for phage glycanase activities. Screening for phage glycanase activities was performed by the method of Eriksson et al. (7). In short, mid-log-phase cells of the uridine diphosphate-galactose-4-epimeraseless mutant, S. typhimurium LT2Ml, were labeled with D-galactose-1-14C (0.5 ,uCi/ml, 2 mg/ml) in their LPS for 3 h. To measure glycanase activities, the 14C-labeled cells, twice washed in PBS, were mixed with dilutions of the phage to be tested. After 60 min of incubation at 37°C, cells were sedimented at 2,000 x g for 20 min in the cold, and portions of the supernatant were withdrawn and assayed for 14C activity. Screening for endorhamnosidase activities. To test whether a specific phage glycanase possessed the ability to cleave the a-L-rhamnosyl linkages of the S. typhimurium SH4809 LPS, the dialyzable crude oligosaccharide preparation (see below) was tested for its inhibitory activity on Bandeiraea simplicifolia lectin agglutination of human type B erythrocytes. Inhibition 50% or more of that obtained with melibiose (calculated on a molar basis assuming a mean molecular weight of the crude oligosaccharide of -1,200) was considered as positive.
S. TYPHIMURIUM PHAGE ENDORHAMNOSIDASES
VOL. 32, 1979
Electron microscopy. The preparation to be examined was stained with uranyl acetate by the following procedure: a droplet of the preparation was placed on a grid (200-mesh copper with carbon-shadowed Parlodion film), and excess fluid was sucked off with filter paper. A drop of freshly prepared 1% (wt/vol) uranyl acetate was then applied to the grid. After a few minutes, excess fluid was again removed by touching the grid to the corner of an adsorbent filter paper. The preparations were examined in a Philips EM-200 electron microscope. The estimation of phage dimensions was made by comparison with a calibration grid. Methylation analyses. Methylation analyses were performed as described earlier (14). The oligosaccharides were reduced with sodium borohydride, and after methylation and subsequent work-up, the materials were hydrolyzed. The resulting monosaccharides were transformed into alditol acetates and analyzed by gasliquid chromatography (GLC) and mass spectrometry. For gas-liquid chromatography, a Perkin-Elmer 990 instrument fitted with a 3% OV-225 colunm was used. Gas-liquid chromatography-mass spectrometry was performed with a Varian MAT 311 instrument. Test for phage glycanase hydrolysis of capsular or LPS. The phage to be assayed was mixed at a ratio of 109 to 1010 PFU/mg of polysaccharide in 5 mM ammonium carbonate buffer, pH 7.0, and incubated at 37°C within a dialysis bag immersed in 10 times the volume of the same buffer. After 48 h of incubation, the surrounding dialysis fluid was concentrated to dryness and heated to 50°C under vacuum to remove the remaining ammonium carbonate. The amount of hexose in the reaction mixture and the surrounding
Bacteria
2
4
dialysis fluid was determined by the phenol-sulfuric acid method (5). Miscellaneous methods. Gel chromatography of oligosaccharides was performed on a column (170 by 2.5 cm) of Bio-Gel P-2 (200 to 400 mesh) eluted with water, using trichlorobutanol (0.05%) as the bacteriocidal agent. Separations were monitored by a Waters 403 refractometer, and the fractions were assayed for carbohydrate content by the phenol-sulfuric acid method. For nuclear magnetic resonance recordings, a Jeol FX-100 instrument operated in the PFT mode was used. The spectra were recorded for solutions in D20 at 850C, using extemal tetramethylsilane as standard.
RESULTS
Screening for phage glycanase activity. Twelve phages were selected from a set used for phage typing of clinical isolates of S. typhimurium (15). The lytic spectra of these phages on the reference set of S. typhimurium strains is given in Table 1. The various phages were incubated with formaldehyde-treated S. typhimurium LT2-M1 cells which had been labeled with ['4C]galactose in their LPS (7). After centrifugation, '4C-labeled material was found in all supernatants, indicating that all 12 phages possessed glycanase activities (Table 2). The same batch of ['4C]galactose-labeled LT2-M1 cells was also subjected to hydrolysis by the P22 endorhamnosidase by incubating the cells with
TABLE 1. Lytic spectra of S. typhimurium phagesa 34 33 32 31 30 28B 8
37
+
+ +
+ +
+ +
+b +
+
+
+
+
+
+
+
+
+
+
+
+
+ + + + + +
+ + + +
+ + + +
+ + + +
+
+
+
+
+
2 3 4 5a 6a 6b 7 8 9 10 11 12 13 14 15 16 18 19 20 21 22 23a
+
+
+ +
+ i
+
+b
+ + + + +
+ +
+
+
+
+b + +
+b
+ +
+
+
+ + + +
39
36
+
1
+
585
+
+
+
+ +
+ +
+
+b+
+ +
+b
+
+
+
+b
+b
+
+
+
+
+
+
+b
+b +
+
+
+
+
+
+
+ +b
+
+
+
+ +
+
+
+
a Phages investigated on the standard set of S. typhimurium strains used for phage typing of clinical isolates. +, Sensitivity to phage indicated. b Strain was used for propagation.
586
SVENSON ET AL.
TABLE 2. Glycanase activities of S. typhimurium phages assayed by ["4C]galactose release' Phage Titer % Releaseb 2 5 x 107 10 (4) 4 1.5 x 1010 49 (33) 8 3.2 x 1012 50 (48) 28B 3.1 x 1012 55 (48) 30 6.7 x 1010 43 (48) 31 1 x 109 46 (12) 32 1.6 x 1010 45 (33) 33 1.4 x 1012 64 (48) 34 1.3 x 1010 65 (32) 36 1 x 109 68 (12) 1 x 1010 37 39 (30) 1 x 109 39 54 (12) a 14C-labeled material was released from S. typhimurium LT2-M1 cells. To 100-,ul portions of washed S. typhimurium LT2-M1 cells previously labeled in their LPS with [14C]galactose were added 900-pl amounts of the various phage preparations. After incubation for 1 h at 37°C, cells were sedimented by centrifugation, 500-,ul portions of the supernatants were withdrawn, and the released 14C activity was determined. The same batch of [14C]galactose-labeled S. typhimurium LT-2 M-1 was also assayed against a PEG-purified P22c2 phage stock. Maximal release (48% of total) was obtained at 5 x 1010 PFU per assay. b Values in parentheses were obtained by using equal numbers of PFU of a purified phage P22c2 preparation.
different amounts of a PEG-purified phage P22 stock and the percentage of release compared with those obtained by the 12 phages under investigation (Table 2). In all instances (except phage 30), the crude phage lysates showed higher release activities per PFU than did the purified phage P22 stock. To confirm that the release of 14C-labeled material from the S. typhimurium LT2-M1 cells was due to phage-associated glycanase activities, crude lysates of the respective propagation strains were tested for hydrolytic properties. About 5 x 1011 cells of each strain were lysed by sonication (three times 10 s at 25W in a Labsonic 1510 sonicator), and the crude bacterial lysates were incubated with [14C]galactose-labeled S. typhimurium LT2-M1 cells. None of the bacterial lysates released any 14C-labeled material. In a control experiment, the various phage lysates were, after sonication as described above, found to be equally active as before sonication. The PEG-purified phage 28B and 36 stocks were further purified by density gradient ultracentrifugation. In both cases, the phage-containing fractions displayed the highest release activities of [14C]galactose-labeled material from S. typhimurium LT2-M1 cells. The 50% maximal release (45% of total label was maximally released) values were, for phage 28B and 36, 8.8
J. VIROL.
109 and 1.1 x 108 PFU, respectively. These data indicated that the glycanase activities of phages 28B and 36 were associated with the
x
phage particles. Screening for endorhamnosidase speci-
ficities. In another series of experiments, the various phages were incubated with partially delipidated LPS from S. typhimurium SH 4809 within dialysis sacks immersed in buffer. All 12 phages degraded the LPS to dialyzable oligosaccharides as evidenced by recovery of 30 to 50% of the total carbohydrate in the surrounding dialysis fluid (data not shown). Glycanases cleaving the a-L-rhamnosyl linkages in the S. typhimurium SH4809 LPS (Fig. 1, Table 4) should give rise to oligosaccharides possessing a terminal, nonreducing ca-D-galactosyl group. The agglutination of human type B erythrocytes with the a-D-galactosyl-specific B. simplicifolia lectin was inhibited by the crude oligosaccharide preparations (obtained from the dialysis experiments described above), thus suggesting that all 12 phages exhibited endorhamnosidase activity. Electron microscopic examination of phages. In Fig. 2, the morphology typical for all 12 phages is shown. They all have a head with hexagonal symmetry about 50 nm in diameter to which a short, noncontractile tail carrying six spikes is attached. This appearance is typical for type C phages, based on the classification of Bradley (4). Two of the phages, 28B and 36, were chosen for a more detailed study. Phage 28B was chosen for its narrow lytic spectrum (Table 1) and ease of propagation. Phage 36, however, had a broad lytic spectrum (Table 1) also lysing the S. typhimurium SL3622 (Fig. 1 and Table 4). Furthermore, phage 36 showed a high '4C release activR a
D-Glcp 4/6
a3i
[a-D-Ma1nP-(l1- 4)-a-L-RhaP-(l1- 3)-D-Gaip]n -+2)R
a
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a-D-Manp-(1
4)-a-L-Rhap-(l
3)-a-D-Galp-
(1 -*CORE
LipA
FIG. 1. General structure of LPS from Salmonella serogroups A, B, and Dl. R indicates abequose (3,6dideoxy-D -xylo-hexose) in Salmonella B, or tyvelose (3,6-dideoxy-D -arabino-hexose) and paratose (3,6-dideoxy-D-ribo-hexose) in Salmonella Dl and A, respectively. Broken arrow indicates substitution occurring only in some strains. n, Number of repeating units in 0-chain, varying from 4 to 30.
S. TYPHIMURIUM PHAGE ENDORHAMNOSIDASES
VOL. 32, 1979
- * < =, *S t .--; '! i-
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In %
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as compared with the phage P22 standard (Table 2); i.e., it could be overproducing the glycanase. Isolation of phage 36 subviral elements possessing glycanase activity. Subviral
ity
587
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